New Approach for Targeted Treatment of Mild COVID-19 by Honeysuckle through Network Pharmacology Analysis

Department of Pharmacy, The People’s Hospital of Guangxi Zhuang Autonomous Region, Guangxi, China Institute of Green Algae, Longhua Branch, Shenzhen People’s Hospital, The Second Clinical Medical College of Jinan University, Shenzhen, 518120 Guangdong, China Department of Cardiology, The People’s Hospital of Guangxi Zhuang Autonomous Region, Guangxi, China Department of Pathology, Longhua Branch, Shenzhen People’s Hospital, The Second Clinical Medical College of Jinan University, Guangdong, Shenzhen 518120, China


Introduction
As of 9 July 2021, more than 4 million people had died from coronavirus disease 2019 (COVID-19) [1]. The most common symptoms of COVID-19 included fever, dry cough, and fatigue. However, infected patients may also experience other symptoms, including shortness of breath and difficulty breathing. Most people infected with the virus showed mild to moderate symptoms, but people with underlying conditions such as cardiovascular disease and diabetes were more likely to develop severe COVID-19. Severe acute respiratory syndrome coronavirus 2(SARS-COV-2) was the cause of COVID-19. SARS-COV-2 was a β-coronavirus that caused mild or severe acute respiratory syndrome when the virus infected the respiratory tract. Subsequent releasing of cytokines led to cytokine storms, which may develop into severe COVID-19 [2]. In addition, a large number of data suggested that mild or severe cytokine storm exists in severe patients, which had become an important cause of death [3]. Blocking cytokine storms at the onset of mild symptoms was expected to be a direct new way to treat COVID-19 patients.
Up to now, traditional Chinese medicine (TCM) had played an important role in COVID-19 diagnosis and treatment protocols released across China. Among them, honeysuckle, as a common Chinese medicinal material, often appeared in the prevention and treatment plan of COVID-19 issued by the state as an important part of the common heat-clearing medicine in clinical practice [4].
For example, honeysuckle, as an important antiviral component, had been included in Lianhuaqingwen. This made Lianhuaqingwen showed anti-2019 coronavirus activity [5]. It had been reported that honeysuckle can effectively relieve clinical symptoms of COVID-19 and inhibit SARS-COV-2 replication [6]. In addition, according to the findings of relevant scholars, the combination of honeysuckle and conventional treatment improved lung CT, increased white blood cell count, and reduced C-Reactive protein level. This effect was related to the effect of honeysuckle on immune response and the production of inflammatory factors [7]. Honeysuckle efficacy against lung infection also included immunopathological changes, such as lymphocytopenia and cytokine elevation, which were important drivers of disease progression and death in coronavirus infection [8]. These findings suggested that honeysuckle had great potential for the treatment of COVID-19, including mild COVID-19.
Traditional pharmacological methods were difficult to elucidate the complex mechanism of TCM in treating diseases because TCM had many components and targets. Network pharmacology was a new method emerging in recent years, which integrated the interactions of drugs, targets, pathways, and diseases into a biological network system, revealing the overall comprehensive effects of Chinese medicine prescriptions and their multipath, multicomponent, and multitarget treatment of diseases [9]. Honeysuckle had previously been used to treat viral pneumonia. Therefore, the main effective compounds were screened out in this study, and the mechanism of honeysuckle against mild COVID-19 was gained a new understanding through network pharmacology method and ADMET characteristic analysis. From the perspectives of gene expression and enrichment analysis, the potential pharmacological mechanism of honeysuckle extract in the treatment of mild COVID-19 was explained, providing a new idea for the prevention and treatment of mild COVID-19 with traditional Chinese medicine.

Screening of the Active Components and Targets of
Honeysuckle. The active components of honeysuckle were searched through Traditional Chinese Medicine Database and Analysis Platform (TCMSP, https://old.tcmsp-e.com/tcmsp .php). Oral bioavailability ðOBÞ ≥ 30% and drug similarity ðDL Þ ≥ 0:18 were selected as the screening conditions [10]. The included active components were input into PubChem database to search for the corresponding molecular structure and record the corresponding CID. The active components with CID were paired with potential targets one by one, and the target protein and gene information were corrected by STRING database to obtain the main active components targets of honeysuckle.

Analysis of Pharmacokinetic
Characteristics. The chemical structure SDF file of active components was downloaded from PubChem database to predict the physicochemical properties of components based on SwissADME database (http://www.swissadme.ch/). Compounds with molecular weight (250 g/mol ≤ MW ≤ 350 g/mol) were candidate compounds. The pharmacokinetic properties of candidate compounds were predicted by pkCSM database (http://biosig .unimelb.edu.au/pkcsm/).

Assessment of Immune Cell Infiltration of Mild COVID-19.
CIBERSORT used a data composed of 547 genes to calculate the infiltration ratio (PMID: 25822800). It described each immune cell subtype and accurately quantified the different immune cell compositions using deconvolution algorithms. Expression of matrix were uploaded to CIBERSORT website (http://cibersort.stanford.edu), to assess the infiltration ratio of 22 immune cell. In the results, immune cells with P < 0:05 had statistically significant differences. The Corrplot toolkit calculated correlations between immune cells and genes.

Results
3.1. Honeysuckle Was Safe as a Medicine to Treat Diseases. 236 components of honeysuckle were identified by TCMSP. According to the screening conditions of OB ≥ 30% and DL ≥ 0:18, 13 active components with Pubchem_CID were identified, corresponding to 119 nonrepeating targets ( Table 2). The MW of seven compounds ranged from 250 to 350 g/mol and were identified as candidate compounds ( Figure 1). The 7 compounds had 96 targets. Pharmacokinetic characteristics showed that most of the candidate compounds showed a good oral availability, and all the tested compounds had relatively low steady-state distribution volume. Most had low bloodbrain barrier and central nervous system permeability values. Five compounds did not show any specific toxicity problems ( Figure 2). It was suggested that the toxicity of honeysuckle as medicine was not high, but some components still needed attention.  To study the transcriptional profile associated with mild COVID-19 infection, we first removed the batch effect after the combination of multiple datasets (Figure 3(a)) and performed genome microarray analysis on 17 mild COVID-19 samples and 20 healthy samples. Principal component analysis of the whole gene showed that the two groups formed a unique cluster and were separated from each other (Figure 3(b)). In our microarray analysis, 2399 DEGs were identified, including 1255 downregulated genes and 1174 upregulated genes. There was a significant difference in expression between the two groups, with the maximum multiple of difference greater than 10 (Figures 3(c) and 3(d)). These results revealed not only unique transcriptional characteristics of mild COVID-19 samples compared to healthy samples but also highly differentiated expression patterns between the two.

Honeysuckle Targets and Mild COVID-19 DEGs Were
Significantly Enriched in Multiple Immune Signaling Pathways. In order to determine the main biological process of honeysuckle targeted therapy for mild COVID-19, enrichment analysis of honeysuckle target and mild COVID-19 DEG was conducted, respectively. Mild COVID-19 DEG was enriched in 1099 GO pathways, 138 KEGG pathways, and 527 Reactome pathways (P < 0:05). The results showed that DEGs were mainly involved in a variety of biological processes, including protein binding, ATP binding, DNA binding, positive regulation of transcription by RNA polymerase II, and extracellular space. KEGG analysis showed that DEGs were mainly enriched in metabolic pathways, NOD-like receptor signaling pathway, protein processing in endoplasmic reticulum, and NF-kappa B signaling pathway. Reactome pathway enrichment results showed that DEG was mainly enriched in metabolism of

No.
Compound PubChem_ CID Formula Structure Gene_ID proteins, immune system, cytokine signaling in immune system, and signal transduction (Figure 4(a)). Honeysuckle targets were enriched in 941 GO pathways, 130 KEGG pathways, and 341 Reactome pathways (P < 0:05 ). The GO pathways included protein binding, positive regulation of transcription by RNA polymerase, components of plasma membrane, and ATP binding. Also, such are involved in fluid shear stress and atherosclerosis, small cell lung cancer, IL-17 signaling pathway, arginine biosynthesis, tumor necrosis factor (TNF) signaling pathway, p53 signaling pathway, NF-Kappa B, and other KEGG pathways. The targets were also mainly enriched in immune system, signal transduction, metabolism, cytokine signaling in the immune system, protein metabolism, and other Reactome pathways. The most significant difference was in immune pathways ( Figure 4(b)).

The Number of Natural Killer (NK) Cells and Hematopoietic Stem Cells Decreased in Mild CCOVID-19.
Based on the expression matrix of 30354 genes, the content and proportion of 22 kinds of immune cells in 49 samples were analyzed. The content of immune cells was shown in Figure 7(a), and the proportion of immune cells in each group was significantly different. To identify changes in individual immune cell expression after infection, we observed differences in the proportion of 22 immune cells in the mild COVID-19 group compared to the control group. It turned out that when virus infected the respiratory tract causing mild acute respiratory syndrome: CIBERSORT assessment results showed that T cells CD8, monocyte, dendritic cells resting increased in proportion with relatively high infiltration; natural killer (NK) cells activated, mast cell activated ratio decreased, and relatively high infiltration (Figure 7(b)). TIMER evaluation results showed an increased proportion of myeloid dendritic cells and a relatively high degree of infiltration (Figure 7(c)). XCELL evaluation results showed that the proportion of myeloid dendritic cell activated and T cell regulatory (Tregs) increased, and the infiltration was relatively high; hematopoietic stem cells, monocyte proportion decreased and relatively high infiltration (Figure 7(d)). In general, the expressions of T cells CD8, myeloid dendritic cells, and Tregs were significantly increased in mild COVID-19 infection symptoms. However, it inhibited the expression of hematopoietic stem cells, NK cells, and Mast cells. With the disease progression of COVID-19 patients, the analysis results of this study showed that the number of NK cell cells decreased [13], the number of Tregs increased [14], and hematopoietic stem cell decreased [15], which were consistent with previous research results.
The correlation between the proportion of immune cell infiltration in XCELL results and the expression of five characteristic genes was analyzed based on mild COVID-19 samples. Previous analyses found significant differences in RELA and MAP3K7 expression levels in mild COVID-19 samples (P < 0:05). It was found that RELA, MAP3K7, and NK cell were significantly different and highly positively correlated, while RELA and MAP3K7 were significantly different and highly negatively correlated with hematopoietic stem cells (Figure 8). In other words, the pharmacological action of honeysuckle in the treatment of mild COVID-19 patients was closely related to RELA, MAP3K7, NK cell, and hematopoietic stem cell.

Discussion
Novel coronavirus infections were characterized by damage to the lungs and immune system. Severe infections led to acute respiratory distress syndrome and sepsis and, ultimately, death [16]. In addition, some patients also developed multiple organ damage and dysfunction [17]. However, there was no specific drug or vaccine that can fully treat  Figure 6: Identification of characteristic immune genes. 201 DEGs (a) and 33 honeysuckle targets (b) were identified from four immunerelated pathways (adaptive immune system, cytokine signaling in immune system, immune system, and innate immune system). Five characteristic immune genes were obtained, and their expression in normal and mild COVID-19 samples was analyzed (c, d).
9 Computational and Mathematical Methods in Medicine COVID-19. Part of the reason may be that a single-targeted drug cannot cure a complex disease through a complex biological network. Despite the lack of strong evidence, TCM had good potential to supplement medical services for COVID-19. Honeysuckle had been observed to be effective in helping to cure COVID-19 patients and stop the COVID-19 pandemic. Therefore, we explored new targets and pathways of honeysuckle against mild COVID-19 from the perspective of network pharmacology.
In this study, significant overlap in enrichment pathways between honeysuckle targets and COVID-19 DEG were found. The immune system, as a strong and significant enrichment pathway, played an important role in the pharmacological effects of honeysuckle in the treatment of mild COVID-19. Recent studies had suggested that immune system damage may be associated with higher mortality among COVID-19 patients [16]. For example, patients may present with lung interstitial lymphocytic infiltration, lymphocytopenia, and peripheral blood T cell hyperactivation [18]. Therefore, inflammation and immune response were important for eliminating infection, may have an important impact on the pathogenesis of SARS-COV-2, and may play a role in the clinical spectrum expression of mild COVID-19 disease. It was well known that pharmacokinetics was an important part of evaluating whether a drug was toxic or absorbable during drug development. In our study, Swis-sADME and pkCSM evaluated that most active ingredients of honeysuckle showed good oral availability and no toxicity issues. It indicated the safety and effectiveness of honeysuckle. Therefore, the multipathway and multitarget results of the active components of honeysuckle not only provide a new useful method for studying traditional Chinese medicine but also may prove the rationality of the compatibility of honeysuckle with other traditional Chinese medicine.
Our analysis found specific differences in immune cell infiltration in mild COVID-19 samples. These findings were consistent with the clinical characteristics of infected patients, including reduced NK cells and hematopoietic stem cells. Two significantly underexpressed and statistically significant immunomodulatory genes were identified in mild COVID-19 samples: RELA and MAP3K7. RELA, as a key subunit of NF-Kappa-B, regulated the stability of regulatory T cells in the immune system [19]. Mitogen-activated protein kinase 7 (MAP3K7) encoded transforming growth factor β-activated kinase 1 (TAK1), which regulated several important downstream effectors involved in immune responses [20]. Correlation analysis showed that these two genes had a strong correlation with NK cell and hematopoietic stem cell. These results suggested that the regulation of this number of genes may reflect the characteristics of mild COVID-19 progression. Therefore, these two genes can serve as potential target markers for honeysuckle treatment of mild COVID-19. Of course, the two immunoregulatory genes needed to be validated in larger microarrays.
The immunopathology of COVID-19 was based on dysregulation of innate and cell-mediated immune responses. A number of studies had found that patients infected with SARS-COV-2 had significantly reduced the number of NK cells and showed a functional failure phenotype. In addition, the expression of inhibitory markers in NK cells increased, leading to decreased expression of IFNγ, IL-2, and TNFα [21]. This suggested that NK cells had an inflammatory phenotype in patients with mild COVID-19. When SARS-COV2 entered the body, the innate immune system acted as the first responder to detect viral infection. At this point, the number of NK cells were reduced, and proinflammatory cytokines were produced to inhibit viral replication. However, excessive cytokine release led to excessive inflammation, triggering cytokine storms that can lead to serious complications of disease [22]. In addition, hematopoietic stem cells (HSC) can differentiate into immune cells, providing defense against viral infection. SARS-Cov-2 binded to the angiotensin converting enzyme 2 (ACE2) expressed in HSC, which not only blocked the proliferation and differentiation of HSC but also led to the overactivation of Nlrp3 inflammasome, which ultimately became the culprit of "cytokine storm" [15]. Thus, the normal differentiation of HSC helped suppress COVID-19 infection. Cytokine storms was often thought to be the cause of COVID-19 cases. In our study, we found that the immune genes RELA and MAP3K7 were strongly correlated with the cytokine storm triggered by NK cells and HSC. Therefore, we believed that in mild COVID-19, honeysuckle extract may increase the infiltration ratio of NK cells/HSC through targeted upregulation of RELA/MAP3K7 expression, and finally inhibited the release of proinflammatory cytokines to achieve the effect of alleviating symptoms, hoping to stifle the disease at the mild stage. In addition, RELA and MAP3K7 may be promoting markers of NK cells, but the specific mechanisms remain to be explored.
In summary, honeysuckle was effective in the treatment of mild COVID-19. But there were still some shortcomings in our study, such as the lack of in-depth research on predicting individual components and key targets and pathways, which required further validation in vivo and in vitro. However, this study confirmed that network pharmacology can be useful in  Figure 8: Correlation between the proportion of immune cell infiltration and the expression of five characteristic genes in XCELL results ( * P < 0:05; * * P < 0:01; * * * P < 0:001).  GSM5019817  GSM5019818  GSM5019819  GSM5019820  GSM5019821  GSM5019822  GSM5019823  GSM5019824  GSM5019825  GSM5019826  GSM5418334  GSM5418377  GSM5418333  GSM5418381  GSM5418335  GSM5418382  GSM5418336  GSM5418350  GSM5418392  GSM5418351  GSM5418393   exploring the mechanism of action of honeysuckle extract in the treatment of COVID-19 with time and cost savings. This study also provided possible candidate components of honeysuckle and related immune genes with potential therapeutic effects on COVID-19, which will provide evidence and new insights for further research on the treatment of COVID-19 by honeysuckle.

Conclusion
In general, it was critical to diagnose an infection in the first stage, before the cytokine storm begins. The cytokine storm triggered by NK cells and HSC leaded to a vicious cycle that eventually led to systemic inflammation and multiple organ failure. If not properly controlled, mild COVID-19 will progress to moderate or even severe COVID-19. Therefore, our findings not only improve our understanding of the pathogenesis of mild COVID-19 but also may provide potential therapeutic targets for honeysuckle treatment of the disease.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.